The main study of the work reported concerned the interaction of Adenovirus type 5 penton antigen with HeLa cells. It was found that penton antigen inhibited the spreading of HeLa cells on glass and this effect, together with the Early Cytopathic Effect or Toxic Effect of the antigen. was used as an indicator of the antigen interaction with cells. Preparations of penton antigen were obtained by fractionation of adenovirus type 5 harvests on diethylaminoethyl Sephedex. It was shown that the inhibition of cell spreading was specific to the penton antigen and that inhibitory activity resided in the base of the antigen particle. as does activity leading to the Early Cytopathic Effect. Two techniques which determined the inhibition of spreading of single HeLa cells were developed and these were used for quantitative and other studies on the antigen/cell interaction. The inhibition of spreading of single Meta cells by antigen as found to follow first order kinetics. and the dose/response relationship indicated that antigen particles acted singly without cooperation. Kinetic and adsorption experiments showed that little or no antigen activity was lost In bringing about the inhibition of spreading. It was found that attachment of antigen and the development of' resistance to the protective effect of antiserum and trypsin by antigen/ cell complexes were necessary for inhibition of spreading to take place. iii These processes also followed first order kinetics and were uninfluenced by treatment of cells with Sodium fluorides it was suggested that the rates of inhibition and of these intermediate stages ware governed directly by the rate of attachment of antigen to cells. Inhibition of spreading was temperature dependent but not dependent on cellular mecromolecular synthesis. Penton antigen did not affect cellular protein synthesis, cellular giycolysis, or gross functions of the cell membrane. It was confirmed that cells recovered from inhibition of spreading It was concluded that the penton antigen of adenovirus type 5 brought about the inhibition of cell spreading and the Early Cytopathic Effect by cell changes independent of the organisation of the cell responsible for macromolecular synthesis and of membrane function. and that at least some of the processes leading to these cell changes take place at the cell surface.

Bordetella pertussis is the causative agent of whooping cough (or pertussis) that has become resurgent worldwide. Resurgence has been linked to the introduction of acellular pertussis vaccines (ACVs) and the evolution of B. pertussis away from vaccine-induced immunity. In 2012, the United Kingdom suffered a major pertussis outbreak. I conducted whole genome sequencing and genomic analysis of 95 strains including those isolated from the UK outbreak and demonstrated that although large-scale genetic changes in B. pertussis were not the cause of this outbreak, vaccine-antigen encoding genes are evolving at higher rates than other surface protein-encoding genes, this difference becoming more pronounced since the introduction of the ACV in the UK. The dramatic increase in the frequency of pertactin (Prn)-deficient strains worldwide is possibly in response to vaccine-mediated selection pressure. However, just one Prn deficient strain was identified among the UK outbreak strains. Prn expression and IgG binding to B. pertussis obtained with post-vaccine sera was determined by flow cytometry and compared between pre-outbreak and outbreak strains, but no significant differences were observed. However, a positive correlation between Prn expression and post vaccination induced IgG binding to strains was identified, supporting the idea that strong immunological selection pressure is exerted on Prn and this is playing a role in the evolution of B. pertussis. The rapid evolution of vaccine-antigen encoding genes raises serious concerns regarding the ability of current vaccines to control pertussis.

Tissue damage initiates the release of a complex, interacting collection of chemical signals. The coordinated function of these signals gives rise to an inflammatory event, whereby circulating immune cells are recruited to clear pathogens. Invertebrate models of tissue damage have revealed a key role for damage-associated signals, specifically hydrogen peroxide (H2O2), in attracting immune cells to sites of tissue damage. The Src family kinase (SFK), Lyn, is oxidised by H2O2 in zebrafish wound models, and subsequent activation of Lyn triggers directed cell motility. In mammalian systems, H2O2 is an important second messenger, however, its role as a damage signal and its association with SFK signalling remains unclear. The aims of this thesis are to investigate how immune cell function and migration in response to damage-associated signals transfers into other models. To address this, we have used an in vivo Drosophila melanogaster embryonic wounding model and in vitro assays of human innate cell function and migration. The migration of Drosophila hemocytes in response to a wound was impaired in embryos lacking functional Src42A or Shark kinase. However, hemocyte motility was impaired in embryos bathed in exogenous H2O2. In vitro, H2O2 inhibited human innate cell motility, chemotaxis, actin reorganisation and phagocytosis, but activated intracellular signalling pathways and did not affect receptor expression or cell viability. Exogenous ATP activated chemokinesis and rapid actin reorganisation. The in vitro effects of immune-related ligands were inhibited by pharmacological inhibition of SFK, Syk, and PI3K signalling. In particular, inhibition of class IA PI3K isoforms p110β and p110δ, but not p110α, disrupted monocyte MCP-1-mediated actin reorganisation and spreading. SFKs are required for Drosophila immune cell migration to a wound and for human innate cell migration to chemoattractants. While endogenous ROS production is important for immune cell function, exogenous H2O2 may negatively modulate downstream mediators of chemokine signalling. H2O2 and ATP are distinct in their abilities to activate immune cells and initiate chemokinesis. Intracellular kinases regulate basal and chemoattractant-mediated motility and are therefore attractive targets for therapeutic management of inflammatory disease.

No description available.

Dendritic cells (DCs) are the watchmen of the immune system. They survey the peripheral tissues and display samples of surrounding antigens for inspection by T cells. Pathogenic or inflammatory signals trigger DCs to mature, upregulating their expression of MHC and costimulatory molecules and converting them into potent T cell stimulators. The character of the activated DC depends not only on the stimulus but also on concomitant environmental signals. This thesis tests the hypothesis that in vitro manipulation of DC function can be used to direct immune responses in vivo. The work focuses on the cytokine IL-10 and asks whether its impact on DC maturation can mediate T cell tolerance. IL-10 has been reported to trap DCs in an immature state, leading to antigen presentation without full costimulation and consequent T cell anergy. Data presented here show that DCs do become activated in the presence of IL-10. They downregulate antigen uptake and, 6 hours after stimulation, display high levels of MHCII and B7. Their activation is short-lived, with both MHC and B7 expression returning to baseline within 18h. Even at 6 hours, with high levels of surface MHC and costimulation, the IL-10 treated DCs express little 1L-12 and fail to elicit strong T cell proliferation. IL-10 seems not to act by inhibiting DC maturation but instead by dictating the kinetics and quality of their activation. The consequence of this DC activation for the responding T cells is also examined. Both in vitro and in vivo, using co-cultures and adoptive transfers of TCR transgenic T cells followed by DC-based immunisation, initial contact with IL-10 treated DCs appears to leave T cells hyporesponsive to subsequent challenge. In a mouse model of autoimmunity, these DCs suppress disease. Taken together these data suggest that, rather than preventing DC maturation, IL-10 directs an active DC phenotype that can regulate immune responses. These DCs have exciting therapeutic potential.

A kidney transplant is the optimum treatment for patients with renal failure, both prolonging life and improving its quality (Parfrey, 2000. Wolfe, 1999). Transplanting an organ subjects it to Ischaemia reperfusion injury (IRI) as the vascular supply is temporarily disrupted and then reinstated. IRI is a risk factor for delayed graft function (DGF) (Bronzatto, 2009), which prejudices both short and long-term graft survival (Ojo, 1997). The severity of IRI has also been linked with early changes within the renal microvasculature that correlate with subsequent impaired organ function and DGF following transplantation (Schmitz, 2008). At the current time there are no specific treatments for IRI. Heme oxygenase-1 (HO-1) is a 32kDa enzyme that catalyses the breakdown of Heme molecules to Biliverdin, Carbon monoxide and free Iron. Studies suggest that induction of HO-1 prior to surgery may be beneficial by reducing the severity of injury in animal models of IRI and transplantation (Amersi, 1999. Tullius 2002). The majority of such studies utilise heavy metal protoporphyrins as HO-1 inducing agents. These substances are highly toxic, preventing their use in clinical practice. Heme arginate (HA) is a clinically licensed drug used in humans for the treatment of porphyria. It is well tolerated, with a minimal side effect profile (Mustajoki, 2003). It has also been shown that HA can induce HO-1 in healthy human volunteers (Doberer, 2010). The principal aims of this thesis are to determine whether the administration of HA can confer protection within in vitro and in vivo models of IRI and renal isograft transplantation. I assess the structural integrity of the microvasculature and evaluate changes in the inflammatory cell populations following transplantation and IRI to determine whether these correlate with the degree of renal tubular injury observed. Initial In vitro experiments demonstrate the potential for HA to induce HO-1 in a murine cardiac endothelial cell line (MCEC-1). I subsequently develop and characterise an in vitro model that simulates the changes in gaseous tensions encountered during IRI in vivo, and show that MCEC-1 cells that are pre-treated with HA are significantly protected against exposure to these adverse conditions (n=3, p < 0.05). In subsequent in vivo experiments, HA administration was shown to up-regulate functional HO-1 within murine renal tissue. Mice that were pre-treated with HA 24 hours prior to surgery showed significant preservation of renal tubules and renal function (creatinine) in a murine model of renal IRI (n=8, p < 0.05). In a series of murine renal isograft transplants, HA pre-treatment of organ donors resulted in significant protection of renal tubules against IRI (n=10, p < 0.05). An apparent, but statistically insignificant, trend toward protection of renal tubules was also observed in organ recipients that were pre-treated with HA (n=10, p=0.21). Further experiments are necessary to clarify the underlying mechanisms responsible for the apparent protective effects of HA against tubular injury in vivo.

The tools and techniques for the study of porcine T-cells lag behind what is currently attainable in human T-cells, so this thesis was initially focused on improvements in this field. This study established long-term culture of porcine T-cells, T-cell clone procurement and relevant T-cell assays. These techniques were then used to investigate cytotoxic T-cell responses to Influenza A virus (IAV) in pigs. IAV is highly mutative and novel strains can be generated following reassortment between different viral strains. IAV is endemic in the global pig population and in some circumstances the virus can pass between humans and pigs and other animals. Pigs can therefore, potentially be a source for the generation of new and possibly pandemic influenza strains. The risk this poses to global human health, together with the negative effects of IAV infection within pig herds, highlights the need to improve our knowledge of IAV in pigs. This study identified four new MHC class I restricted IAV epitopes, derived from the viral nucleoprotein. Cytotoxic T-cells recognising these IAV epitopes were detected at high numbers ex vivo in samples from vaccinated pigs. The structures of these IAV epitopes in complex with their respective MHC class I molecules were resolved and revealed the primary anchor positions within the peptides. This enabled peptide binding motifs to be defined for two porcine MHC-I alleles. These peptide binding motifs can be utilised for efficient epitope prediction. This study also identified super-agonist ligands for two of the MHC-I restricted IAV epitopes. Overall, this work has opened up the study of porcine T-cells to a level previously unattainable and has contributed to our knowledge of IAV in pigs. It has paved the way for further experiments investigating IAV in pigs, other porcine diseases and for using pigs as an animal model for human disease.

In contrast enhanced ultrasound images (CEUS) that use microbubbles, nonlinear propagation of ultrasound creates artefacts which significantly impact the qualitative and quantitative assessments of tissue perfusion. Such artefact originates from tissue reflecting/scattering nonlinearly propagated ultrasound pulse. Consequently such tissue is misclassified as microbubbles which also generate nonlinear signals. This thesis reports the development and evaluation of an algorithm to reduce the nonlinear propagation artefact in CEUS. The method was evaluated in simulations, and on in vitro and in vivo data at both high and low ultrasound frequencies. Ways to further improve the performance of the method were also investigated. Firstly, the artefact correction algorithm was developed. The algorithm makes use of two independent datasets that are acquired simultaneously during CEUS; the Bmode image, which is dominated by tissue information, and the contrast specific image, which contains information on blood (signal) due to microbubbles, but confounded with some amount of tissue signals (artefact). The unwanted tissue contribution of the contrast specific image is reconstructed by estimating the two components that make up this contribution, namely, the underlying tissue distribution and the nonlinear point spread function (PSF) of the imaging system. To initially evaluate the algorithm, a simulation platform was developed to study artefact generation at various Mechanical Indices (MI), microbubble concentrations and frequencies. The algorithm was then evaluated using the simulation data. The results show that the algorithm is able to reduce the nonlinear propagation artefact at different MI, concentration and frequency under both ideal and noisy conditions. Next, artefact correction was applied to carotid artery imaging. The performance of the algorithm was evaluated using flow phantoms with large and small vessels containing microbubbles of various concentrations at different acoustic pressures. The algorithm significantly reduces nonlinear artefacts while maintaining the contrast signal from bubbles to increase the contrast to tissue ratio (CTR) by up to 11 dB. Contrast signal from a small vessel of 600 µm in diameter buried in tissue artefacts prior to correction is recovered after the correction. The algorithm was then evaluated using in vivo CEUS data acquired on patients’ carotid arteries. The algorithm is able to increase the CTR at the far-wall by up to 7.4 dB in vivo. Artefact correction was then improved by taking the spatial variance of the ultrasound field into account and improving the nonlinear PSF estimation. The new version of the algorithm was tested on in vitro and in vivo data and the improvements verified. The new version of the algorithm provides an additional increase in CTR by up to 5.4 dB in the far field, 4.3 dB at focus and 3.2 dB in the near field for the in vitro data over previous results. The additional increase in CTR for the in vivo data is up to 4 dB more in the near field and 5 dB more in the far field over the previous results. Nonlinear propagation correction was also applied to deep tissue imaging where lower ultrasound frequency than carotid imaging was used. The algorithm could suppress tissue signal more than 6 dB. However, due to the strong presence of the microbubbles in the B-mode image at low frequencies, the algorithm reduces microbubble signal by up to 2 dB. The resulting increase in CTR is up to 4 dB under specific imaging conditions. However, depending on the imaging geometry and acquisition settings used, it could fail to produce an increase in CTR. A possible future direction is to combine the algorithm with an attenuation correction method to improve perfusion quantification. The clinical efficacy of the combined nonlinear propagation and attenuation correction could be evaluated. Given that the method is purely post-processing, it is easier to implement it on current commercial scanners than some other existing techniques. The implementation of the algorithm using GPUs could be investigated and could possibly instigate translation into clinics.

Endoscopy is a standard procedure for visualising the human gastrointestinal tract. With the advances in biophotonics, imaging techniques such as narrow band imaging, confocal laser endomicroscopy, and optical coherence tomography can be combined with normal endoscopy for assisting the early diagnosis of diseases, such as cancer. In the past decade, optical biopsy has emerged to be an effective tool for tissue analysis, allowing in vivo and in situ assessment of pathological sites with real-time feature-enhanced microscopic images. However, the non-invasive nature of optical biopsy leads to an intra-examination retargeting problem, which is associated with the difficulty of re-localising a biopsied site consistently throughout the whole examination. In addition to intra-examination retargeting, retargeting of a pathological site is even more challenging across examinations, due to tissue deformation and changing tissue morphologies and appearances. The purpose of this thesis is to address both the intra- and inter-examination retargeting problems associated with optical biopsy. We propose a novel vision-based framework for intra-examination retargeting. The proposed framework is based on combining visual tracking and detection with online learning of the appearance of the biopsied site. Furthermore, a novel cascaded detection approach based on random forests and structured support vector machines is developed to achieve efficient retargeting. To cater for reliable inter-examination retargeting, the solution provided in this thesis is achieved by solving an image retrieval problem, for which an online scene association approach is proposed to summarise an endoscopic video collected in the first examination into distinctive scenes. A hashing-based approach is then used to learn the intrinsic representations of these scenes, such that retargeting can be achieved in subsequent examinations by retrieving the relevant images using the learnt representations. For performance evaluation of the proposed frameworks, extensive phantom, ex vivo and in vivo experiments have been conducted, with results demonstrating the robustness and potential clinical values of the methods proposed.

The applications and techniques of X-ray fluoroscopy are outlined, and the advantages to be gained from intensification of the fluoroscopic image are indicated. Existing intensification systems are discussed and compared with the requirements of the ideal system. The possibility of fulfilling these requirements with a fluorescent screen optically coupled to a high gain image intensifier is examined. The desirability of television presentation of the final image is shown. Construction of an intensification system incorporating a transmitted secondary electron multiplication image intensifier and cathode potential stabilised Emitron television camera is described. Tests of the component parts of the apparatus are described, and on the basis of these results a theoretical estimate of the performance of the complete system is obtained. Practical tests are described which show that considerable benefits can be obtained by the use of the system. The performance of the image intensifier-television camera combination as a detector of weak optical images is indicated. Methods of improving the performance of the system are discussed, leading to the conclusion that an improved image intensifier is desirable. Previous designs of cascade type image intensifiers are outlined, and the design and construction of a tube of this type suitable for the X-ray system are detailed. The practical performance of the cascade tube developed is examined, and results obtained when using it in the X-ray intensification system are presented.